Two molecular forms of alpha-lactalbumin with apparent molecular weights of 21,800 (alpha-LAL) and 22,500 daltons (alpha-LAH) were separated from rat milk. The alpha-Lactalbumins were purified to homogeneity by gel filtration with Sephadex G-150, followed by DEAE-cellulose chromatography. Both alpha-lactalbumins are glycoproteins. Alpha-LAL has one residue of glactose and one residue of hexosamine more than alpha-LAH. Alpah-LAH has one lysine, one glutamic acid and four proline residues more than alpha-LAL. The two alpha-LA species can be discriminated by rat or cow galactosyltransferase in a lactose synthase assay, but are antigenically similar in a radioimmunoassay.